Whereas UDP-glucuronosyltransferase (UGT) isozymes detoxify numerous endogenous and exogenous chemical toxins present in our daily diet and environment via linkage to glucuronic acid, the special properties and enzymatic mechanism(s) that enable the endoplasmic reticulum (ER)-bound isozymes to convert innumerable structurally diverse lipophiles, including therapeutics, to excretable glucuronides are unknown. We have accumulated evidence that regulated phoshorylation of UGTs is critically important for their activity and evidently their substrate selections. Inhibition of in-cellulo UGT activities by treatment with various kinase inhibitors, incorporation of 33P orthophosphate into immunoprecipitable UGT proteins and loss of UGT activities when protein kinase C (PKC) and/or tyrosine kinase (TK) sites are mutated provide evidence that all UGTs undergo required phosphorylation. Studies indicate family 1A isozymes require PKC-dependent phosphorylation. Co-immunoprecipitation, co-localization via immunofluorescence and cross-linking demonstrate UGTs and PKC isozymes, functionally, share compartment(s) or complex structure(s). Moreover, substrate and pH changes following mutation of PKC sites in UGT1A7 indicate phosphorylated serine residues participate in substrate selection. On the other hand the critically important family 2B isozymes, UGT2B7 and UGT2B15, which detoxify endogenous genotoxic catechol-estrogens and dysfunctional-prostate-causing dihydrotestosterone (DHT), respectively, require tyrosine phosphorylation with 2B15 also requiring serine/threonine phoshorylation. Src-TK phosphorylates UGT2B7 based on: (a) loss of activity for either Y236F- or Y438F-UGT2B7 mutant, but not for either of three PKC-sites mutants, (b) SrcTK pull-down with anti-UGT, which was abolished with Y438F-UGT2B7, (c) enhancement of recombinant 2B7 activity and Y438-phosphorylation status by co-transfection with wild-type or activated Src, but not dominant-negative Src, (d) crosslinking of 2B7 and Src, which was prevented by pretreatment with Src-specific inhibitor, PP2, and (e) increased activity of affinity-purified UGT2B7 expressed in Src/Yes/Fyn (SYF)-/- cells following its in-vitro phosphorylation by Src. Moreover, 2B7 activity and its Y438 phosphorylation are associated with active Src and not total Src in 8 pairs of normal/tumor breast tissue sets, suggesting 2B7 capacity to detoxify catechol estrogens is dependent upon active Src in breast tissues. Contrariwise, DHT-metabolizing 2B15 exhibited highest activity following expression in SYF-/- cells, but exhibited progressively less activity following expression in SYF+/- and SYF+/+ cells, suggesting Src inhibits 2B15 activity. Evidence that signaling events regulate phosphorylation of PKC-dependent UGT1A isozymes is based on: (a) reversible downregulation of 1A activities treated with both a protein-synthesis inhibitor and a highly-specific PKC inhibitor such as translocation antagonist peptides or SiRNAs and (b) dramatic up-regulation of curcumin-inhibited 1A activities by specific PKC agonists such as DAG, PS and PMA. Additionally, catalase and herbimycin-A inhibition of constitutive or hydrogen peroxide-activated UGTs demonstrated endogenous ROS-related oxidants are behaving as second messengers to maintain PKC-dependent signaling and UGT phosphorylation to support the on-going glucuronidation process. In order to isolate UGT1-kinase(s) complex(es) containing signaling element(s), ER-bound UGT was solubilized with zwitterionic-CHAPS- and sugar-containing BOG-detergents, which stabilized UGT1A7 activity as a complex composed of chaperone phospho-serine/threonine binding protein, 14-3-3, UGT1A7 and PKCepsilon compared to loss of S162A-UGT1A7 activity due to its mutant 14-3-3 binding site. Also, determination of the common donor substrate, UDP-glucuronic acid, binding site(s) present in all UGTs, which was begun earlier by a college student using computer modeling with a special software package, was completed. Computer modeling in our laboratory identified analogous strucuturally-solved UDP-glucose isomerase protein (ID:1XEL in the PDB) that predicted N-292, K-314, K315, and/or K-404 as potential UDP-glucuronic acid binding sites in UGT1A10. Mutational studies in conjunction with recently available affinity probe, 5N3 32PUDP-Glucuronic acid, revealed both UGT1A10 and 1A7 have both a high and low affinity binding site by Scatchard analysis and that K-314 is the critical residue based on resolution of chemically-digested UGTs and their mutants separated in a discontinuous Tricine-SDS polyacrylamide gel system. Finally, we exploited UGT phosphorylation requirement to demonstrate glucuronidation can markedly compromise drug efficacy due to premature clearance and to determine whether inhibition of glucuronidation prior to administration of glucuronidatable drugs will lead to a significant increase in free drug uptake. After having established in-cellulo that mouse recombinant UGTs also undergo phosphorylation, we pretreated antigen-stimulated mice with curcumin to target UGT phosphorylation before administration of immunosuppressant, mycophenolic acid (MPA). Reversible, in-vivo downregulation of UGT phosphorylation caused a 6- to 8-fold increase in both free MPA in blood and immunosuppression. Widely used MPA for renal transplant patients demonstrates glucuronidation can markedly compromise drug efficacy and that transient downregulation of UGT phosphorylation can have a significant effect on efficacy of glucuronidatable drugs.